Isolation of Active Regulatory Elements from Eukaryotic Chromatin Using FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)
ABSTRACT The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). We also provide protocols for different methods of detecting FAIRE-enriched DNA, including use of PCR, DNA microarrays, and next-generation sequencing. FAIRE works on all eukaryotic chromatin tested to date. To perform FAIRE, chromatin is crosslinked with formaldehyde, sheared by sonication, and phenol–chloroform extracted. Most genomic DNA is crosslinked to nucleosomes and is sequestered to the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosome-depleted regions of the genome. The isolated regions are largely coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, enhancers, insulators, and active promoters. Given its speed and simplicity, FAIRE has utility in establishing chromatin profiles of diverse cell types in health and disease, isolating DNA regulatory elements en masse for further characterization, and as a screening assay for the effects of small molecules on chromatin organization.
- SourceAvailable from: Carsten Carlberg
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- "Like most other transcription factors, VDR competes with the intrinsic repressive nature of chromatin for access to its genomic binding sites  . Open chromatin regions can be detected by the genome-wide method formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq)   . "
ABSTRACT: Vitamin D3 belongs to the few nutritional compounds that has, via the binding of its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) to the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. The relation of thousands of genomic VDR-binding sites to a few hundred primary 1,25(OH)2D3 target genes is still largely unresolved. We studied chromatin domains containing genes for the adhesion molecules CD97 and LRRC8A, the glucose transporter SLC37A2 and the coactivator NRIP1. These domains vary significantly in size (7.3 to 956 kb) but contain each one major VDR-binding site. In monocytic cells these four sites are associated with open chromatin and occupied by VDR, while in macrophage-like cells only the sites of LRRC8A, SLC37A2 and NRIP1 are accessible and receptor bound. The VDR site of CD97 does, in contrast to the three other loci, not carry any DR3-type binding sequence. CD97, LRRC8A, SLC37A2 and NRIP1 are early responding 1,25(OH)2D3 target genes in monocytic cells, while in macrophage-like cells they respond less and, in part, delayed. In primary human peripheral blood mononuclear cells from 71 prediabetic subjects of a vitamin D3 intervention study (VitDmet) CD97, LRRC8A, SLC37A2 and NRIP1 can be used as transcriptomic biomarkers for classifying human individuals for their possible benefit from vitamin D3 supplementation. In particular, NRIP1 exceeds the potential of the previously identified marker CD14 by more than 40% and seems to be a well-suited molecular marker for the vitamin D3 status in the hematopoietic system.The Journal of nutritional biochemistry 04/2014; DOI:10.1016/j.jnutbio.2014.04.002 · 4.59 Impact Factor
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- "In contrast to a recent study showing that SIRT7 deacetylates H3K18 at promoters of ELK4 target genes (Barber et al., 2012), RNAi-mediated depletion of SIRT7 did not affect H3K18 acetylation at rDNA (Figure S2B), suggesting that SIRT7 deacetylates different substrates in a gene-specific context. Moreover, Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE) assays which measure nucleosome density according to extractability of crosslinked chromatin (Giresi and Lieb, 2009) revealed no changes in rDNA chromatin compaction, supporting the notion that SIRT7 activates transcription by targeting the Pol I transcription apparatus rather than by changing the chromatin structure (Figure 2G). "
ABSTRACT: Sirtuins are NAD(+)-dependent protein deacetylases that connect metabolism and cellular homeostasis. Here we show that the nuclear Sirtuin SIRT7 targets PAF53, a subunit of RNA polymerase I (Pol I). Acetylation of PAF53 at lysine 373 by CBP and deacetylation by SIRT7 modulate the association of Pol I with DNA, hypoacetylation correlating with increased rDNA occupancy of Pol I and transcription activation. SIRT7 is released from nucleoli in response to different stress conditions, leading to hyperacetylation of PAF53 and decreased Pol I transcription. Nucleolar detention requires binding of SIRT7 to nascent pre-rRNA, linking the spatial distribution of SIRT7 and deacetylation of PAF53 to ongoing transcription. The results identify a nonhistone target of SIRT7 and uncover an RNA-mediated mechanism that adapts nucleolar transcription to stress signaling.Molecular cell 11/2013; 52(3):303-13. DOI:10.1016/j.molcel.2013.10.010 · 14.46 Impact Factor
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- "FAIRE analysis was performed according to the protocol published by Giresi et al. . In short, 18 × 10 6 THP-1 cells were cross-linked identically as for ChIP. "
ABSTRACT: The signaling cascade of the transcription factor vitamin D receptor (VDR) is triggered by its specific ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study we demonstrate that in THP-1 human monocytic leukemia cells 87.4% of the 1,034 most prominent genome-wide VDR binding sites co-localize with loci of open chromatin. At 165 of them 1α,25(OH)2D3 strongly increases chromatin accessibility and has at further 217 sites weaker effects. Interestingly, VDR binding sites in 1α,25(OH)2D3-responsive chromatin regions are far more often composed of direct repeats with 3 intervening nucleotides (DR3s) than those in ligand insensitive regions. DR3-containing VDR sites are enriched in the neighborhood of genes that are involved in controling cellular growth, while non-DR3 VDR binding is often found close to genes related to immunity. At the example of six early VDR target genes we show that the slope of their 1α,25(OH)2D3-induced transcription correlates with the basal chromatin accessibility of their major VDR binding regions. However, the chromatin loci controlling these genes are indistinguishable in their VDR association kinetics. Taken together, ligand responsive chromatin loci represent dynamically regulated contact points of VDR with the genome, from where it controls early 1α,25(OH)2D3 target genes.Biochimica et Biophysica Acta 10/2013; DOI:10.1016/j.bbagrm.2013.10.003 · 4.66 Impact Factor